The coenzyme A requirement of mammalian fatty acid synthetase: evidence for involvement in the elongation of acyl-enzyme thioesters

We have confirmed that coenzyme A is required for rat fatty acid synthetase activity (T. C. Linn, M. J. Stark, and P. A. Srere, 1980, J. Biol. Chem.255, 1388–1392). When rat liver or mammary gland fatty acid synthetase was assayed in the presence of a CoA-scavenging system such as ATP citrate lyase, almost complete inhibition of fatty acid synthesis was observed. The inhibition was reversed by addition of CoA or pantetheine, but not by addition of N-acetylcysteamine or other thiols. In the absence of CoA, the rate of elongation of acyl moieties on both native fatty acid synthetase and fatty acid synthetase lacking the chain-terminating thioesterase I component (trypsinized fatty acid synthetase) was reduced 100-fold. All of the palmitate synthesized slowly by the CoA-depleted native multienzyme was released, by the thioesterase I component, as the free fatty acid; only shorter-chainlength acyl moieties remained bound to the enzyme. The acyl-S-multienzyme thioesters formed by the trypsinized fatty acid synthetase in the absence of CoA contained saturated moieties of chain length C₆-C₁₆; addition of CoA promoted elongation of the acyl-S-multienzyme thioesters without release from the enzyme. The transfer of acetyl and malonyl moieties from CoA to the multienzyme, the reduction of S-acetoacetyl-N-acetylcysteamine and S-crotonyl-N-acetylcysteamine, and the dehydration of S-β-hydroxybutyryl-N-acetylcysteamine, reactions catalyzed by the fatty acid synthetase, were not dependent on the presence of CoA. The hydrolysis of acyl-S-multienzyme catalyzed by thioesterase I, the resident chain-terminating component of the fatty acid synthetase, and thioesterase II, a monofunctional mammary gland chain-terminating enzyme, was also independent of CoA availability as was hydrolysis of an acyl-S-pantetheine pentapeptide isolated from the multienzyme. On the basis of these observations we conclude that CoA is required for the elongation of acyl moieties on the fatty acid synthetase but not for their release from the multienzyme.     

Photoreversible Modulators of Escherichia coli β-Galactosidase. 1-Benzoyl-1-cyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene and 1,1-Dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene

β-Galactosidase (EC 3.2.1.23) is known to be inhibited by some thiol reagents. 1-Benzoyl-1-cyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (1) was shown to be an irreversible inhibitor, while 1, 1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (2) was demonstrated as a positive irreversible modulator causing a rise of up to 186% in β-galactosidase activity. Compound 2 is, however, an irreversible inhibitor of the cysteine proteinase papain (preceding paper). Kinetic values of β-galactosidase at pH 8.3 with o-nitrophenyl β-D-galactopyranoside (ONPG) as the substrate and for compounds 1 and 2 were determined and in view of model experiments, it was assumed that both compounds possibly reacted with the thiol side chain of Cys in the active site inducing allosteric changes in the enzyme. Since the enzyme, modified by compound 1 or 2, was a 2-nitrobenzyl derivative, near-UV irradiation resulted in a recovery of up to 91% and a reduction of the enzyme's activity to 90%, respectively.     

Synthesis of 4,6-dideoxy-3-fluoro-2-keto-β-d-glucopyranosyl analogues of 5-fluorouracil, N-benzoyl adenine, uracil, thymine, N-benzoyl cytosine and evaluation of their antitumor activities

The synthesis of the unsaturated 4,6-dideoxy-3-fluoro-2-keto-β-d-glucopyranosyl nucleosides of 5-fluorouracil (6a), N⁶-benzoyl adenine (6b), uracil (6c), thymine (6d) and N⁴-benzoyl cytosine (6e), is described. Monoiodination of compounds 1a,b, followed by acetylation, catalytic hydrogenation and finally regioselective 2′-O-deacylation afforded the partially acetylated dideoxynucleoside analogues of 5-fluorouracil (5a) and N⁶-benzoyl adenine (5b), respectively. Direct oxidation of the free hydroxyl group at the 2′-position of 5a,b, with simultaneous elimination reaction of the β-acetoxyl group, afforded the desired unsaturated 4,6-dideoxy-3-fluoro-2-keto-β-d-glucopyranosyl derivatives 6a,b. Compounds 1c-e were used as starting materials for the synthesis of the dideoxy unsaturated carbonyl nucleosides of uracil (6c), thymine (6d) and N⁴-benzoyl cytosine (6e). Similarly a protection-selective deprotection sequence followed by oxidation of the free hydroxyl group at the 2′-position of the dideoxy benzoylated analogues 9c-e with simultaneous elimination reaction of the β-benzoyl group, gave the desired nucleosides 6c-e. None of the compounds was inhibitory to a broad spectrum of DNA and RNA viruses at subtoxic concentrations. The 5-fluorouracil derivative 6a was more cytostatic (50% inhibitory concentration ranging between 0.2 and 12 μM) than the other compounds.     

Selected gold compounds cause pronounced inhibition of Falcipain 2 and effectively block P. falciparum growth in vitro

A number of structurally diverse gold compounds were evaluated as possible inhibitors of Falcipain 2 (Fp2), a cysteine protease from P. falciparum that is a validated target for the development of novel antimalarial drugs. Remarkably, most tested compounds caused pronounced but reversible inhibition of Fp2 with K_i values falling in the micromolar range. Enzyme inhibition is basically ascribed to gold binding to catalytic active site cysteine. The same gold compounds were then tested for their ability to inhibit P. falciparum growth in vitro; important parasite growth inhibition was indeed observed. However, careful analysis of the two sets of data failed to establish any direct correlation between enzyme inhibition and reduction of P. falciparum growth suggesting that Fp2 inhibition represents just one of the various mechanisms through which gold compounds effectively antagonize P. falciparum replication.     

Functional effects of reducing and carboxymethylating the single disulfide bond in bovine carboxypeptidase A

Reduction of the single disulfide bond in bovine carboxypeptidase A (Cox) and alkylation of the resulting thiols yielded a modified enzyme containing 1.8 carboxymethylcysteine residues per molecule which exhibited 97 and 80% of native esterase and peptidase activities, respectively. Effects of inhibitors and an activator on peptidase activity were similar to those observed with the native enzyme suggesting minimal alteration of the active site. However, unlike the native enzyme, the modified enzyme underwent rapid inactivation above 15 °C. Similar results were obtained on reduction and alkylation of the single allotype, carboxypeptidase A_β^val. In contrast, modification of carboxypeptidase A (Anson) resulted in lower carboxymethylcysteine contents and large losses in enzymatic activity. This difference is interpreted in terms of the lower conformational stability of carboxypeptidase A_γ, the main constituent of carboxypeptidase A (Anson) [Petra and Neurath (1968) Biochemistry8, 2466].     

Effect of hydroxynitrobenzylation of tryptophan-177 on reactivity of active site cysteine-25 in papain

It is known that the enzymatic activity of papain (EC 3.4.22.2) toward α-N-benzoyl-l-arginine p-nitroanilide can be substantially increased by hydroxynitrobenzylation of Trp-177 through reaction of the enzyme with the active site-directed reagent, 2-chloromethyl-4-nitrophenyl (N-carbobenzoxy)glycinate (S.-M. T. Chang and H. R. Horton, 1979, Biochemistry18, 1559–1563). To determine the effect of such hydroxynitrobenzylation on the nucleophilicity of the essential thiol group at the active site of the enzyme, rates of inactivation by S_N2 reactions of Cys-25 with chloroacetamide and chloroacetate and by Michael addition of Cys-25 to N-ethylmaleimide were monitored. The kinetics revealed that, at pH 6.5, the reactivities of the sulfhydryl group of hydroxynitrobenzylated papain with chloroacetamide and with N-ethylmaleimide are 24 and 27% greater than those of the sulfhydryl group of native papain. At pH 7.1, the rate enhancements are 34 and 39%, respectively. These increases in reactivity of Cys-25 as an attacking nucleophile appear to account for the increased catalytic activity of hydroxnitrobenzyl-papain toward an oligopeptide substrate, α-N-benzoyl-l-phenylalanyl-l-valyl-l-arginine p-nitroanilide, and toward an ester substrate, N-carbobenzoxyglycine p-nitrophenyl ester. However, the presence of the hydroxynitrobenzyl reporter group provides substantially greater improvement (250%) in enzymatic efficiency toward α-N-benzoyl-l-arginine p-nitroanilide, apparently by blocking nonproductive binding of this substrate to the enzyme. Fluorescence changes accompanying the various chemical modifications are interpreted in terms of a charge-transfer interaction between the imidazolium ion of His-159 and the indole moiety of Trp-177 in the active form of native papain, which should help to stabilize the catalytically essential mercaptide-imidazolium ion-pair (Cys-25, His-159).     

Synthesis,anti-inflammatory,analgesic, 5-lipoxygenase (5-LOX) inhibition activities,and molecular docking study of 7-substituted coumarin derivatives

In the present study, 7-subsituted coumarin derivatives were synthesized using various aromatic and heterocyclic amines, and evaluated in vivo for anti-inflammatory and analgesic activity, and for ulcerogenic risk. The most active compounds were evaluated in vitro for 5-lipoxygenase (5-LOX) inhibition. Docking study was performed to predict the binding affinity, and orientation at the active site of the enzyme. In vivo anti-inflammatory and analgesic activity, and in vitro 5-LOX enzyme inhibition study revealed that compound 33 and 35 are the most potent compounds in all the screening methods. In vitro kinetic study of 35 showed mixed or non-competitive type of inhibition with 5-LOX enzyme. Presence of OCH₃ group in 35 and Cl in 33 at C6-position of benzothiazole ring were found very important substitutions for potent activity.     

Human erythrocyte hemolysis induced by selenium and tellurium compounds increased by GSH or glucose: a possible involvement of reactive oxygen species

Oxidative stress can induce complex alterations of membrane proteins in red blood cells (RBCs) eventually leading to hemolysis. RBCs represent a good model to investigate the damage induced by oxidizing agents. Literature data have reported that chalcogen compounds can present pro-oxidant properties with potent inhibitory effects on cell growth, causing tissue damage and inhibit a variety of enzymes. In this study, human erythrocytes were incubated in vitro with various chalcogen compounds at 37 °C: diphenyl ditelluride (1), dinaphthalen diteluride (2), diphenyl diselenide (3), (S)-tert-butyl 1-diselenide-3-methylbutan-2-ylcarbamate (4), (S)-tert-butyl 1-diselenide-3-phenylpropan-2-ylcarbamate (5), selenium dioxide (6) and sodium selenite (7) in order to investigate their potential in vitro toxicity. After 6 h of incubation, all the tested compounds increased the hemolysis rate, when compared to control and compound (2) had the most potent hemolytic effect. The addition of reduced glutathione (GSH) or glucose to the incubation medium enhanced hemolysis caused by chalcogen compounds. The thiol oxidase activity of these compounds was evaluated by measuring the rate of cysteine (CYS) and dithiotreitol (DTT) oxidation. DTT and cysteine oxidation was increased by all the compounds tested. The results suggest a relationship between the oxidation of intracellular GSH and subsequent generation of free radicals with the hemolysis by chalcogen compounds.     

Discovery of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides active against efflux-mediated resistant Streptococcus pneumoniae

A series of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides, were discovered, structurally characterized and biologically evaluated. They have shown good antibacterial activity against selected Gram-positive and Gram-negative bacterial strains. These include N″ substituted 9a-(N′-carbamoyl-γ-aminopropyl)- (6a,c), 9a-(N′-thiocarbamoyl-γ-aminopropyl)- (7a,e), 9a-[N′-(β-cyanoethyl)-N′-(carbamoyl-γ-aminopropyl)]- (9a-c, 9g) 9a-[N′-(β-cyanoethyl)-N′-(thiocarbamoyl-γ-aminopropyl)]-derivatives (10d-f) of 5-O-desosaminyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythronolide A (3).Among the synthesized compounds thiourea 7a and urea 9b have shown substantially improved activity comparable to azithromycin (1) and significantly better activity than the 3-decladinosyl-azithromycin (2) and the parent 3-cladinosyl analogues against efflux-mediated resistant S. pneumoniae.     

Thiol S-methyltransferase from rat liver.

The thiol S-methyltransferase from rat liver has been solubilized and prepared in homogeneous form. The enzyme exists in a monomer of M_r 28,000 although enzyme activity is highly unstable with a half-life of 4 days under the best conditions of storage. The reaction requires S-adenosylmethionine as methyl donor but, as is the case with many enzymes active in detoxification, a large variety of lipophilic compounds can serve as acceptors. Acceptor activity is limited to thiols. The naturally occurring hydrophilic thiols, glutathione and cysteine, act neither as substrates nor as inhibitors. The course of the reaction is biphasic with an initial rapid formation of product that is followed by a slower linear rate. The suggestion is offered that this behavior reflects the slow dissociation of an enzyme-product complex composed of enzyme and S-adenosyl-homocysteine.     

Synthesis and acrosin inhibitory activity of substituted 4-amino-N-(diaminomethylene) benzenesulfonamide derivatives

A series of new substituted 4-amino-N-(diaminomethylene) benzenesulfonamides were synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds showed potent acrosin inhibitory activities with compounds 4o and 4p being significantly more potent than the control compound N-alpha-tosyl-l-lysyl-chloromethyl-ketone (TLCK). The compounds provide a new scaffold for the development of acrosin inhibitory agents.     

Stereoselectivity of chalcone isomerase with chalcone derivatives: a computational study

Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into flavonoids. The activity of CHI is essential for the biosynthesis of flavonoids precursors of floral pigments and phenylpropanoid plant defense compounds. In the present study, we explored the detailed binding structures and binding free energies for two different active site conformations of CHI with s-cis/s-trans conformers of three chalcone compounds by performing molecular dynamics (MD) simulations and binding free energy calculations. The computational results indicate that s-cis/s-trans conformers of chalcone compounds are orientated in the similar binding position in the active site of CHI and stabilized by the different first hydrogen bond network and the same second hydrogen bond network. The first hydrogen bond network results in much lower binding affinity of s-trans conformer of chalcone compound with CHI than that of s-cis conformer. The conformational change of the active site residue T48 from indirectly interacting with the substrate via the second hydrogen bond network to directly forming the hydrogen bond with the substrates cannot affect the binding mode of both conformers of chalcone compounds, but remarkably improves the binding affinity. These results show that CHI has a strong stereoselectivity. The calculated binding free energies for three chalcone compounds with CHI are consistent with the experimental activity data. In addition, several valuable insights are suggested for future rational design and discovery of high-efficiency mutants of CHI.

Toward self-adjuvanting subunit vaccines: model peptide and protein antigens incorporating covalently bound toll-like receptor-7 agonistic imidazoquinolines

Toll-like receptor (TLR)-7 agonists show prominent Th1-biased immunostimulatory activities. A TLR7-active N¹-(4-aminomethyl)benzyl substituted imidazoquinoline 1 served as a convenient precursor for the syntheses of isothiocyanate and maleimide derivatives for covalent attachment to free amine and thiol groups of peptides and proteins. 1 was also amenable to direct reductive amination with maltoheptaose without significant loss of activity. Covalent conjugation of the isothiocyanate derivative 2 to α-lactalbumin could be achieved under mild, non-denaturing conditions, in a controlled manner and with full preservation of antigenicity. The self-adjuvanting α-lactalbumin construct induced robust, high-affinity immunoglobulin titers in murine models. The premise of covalently decorating protein antigens with adjuvants offers the possibility of drastically reducing systemic exposure of the adjuvant, and yet eliciting strong, Th1-biased immune responses.     

Design,synthesis and molecular docking of new N-4-piperazinyl ciprofloxacin-triazole hybrids with potential antimicrobial activity

New N-4-piperazinyl ciprofloxacin-triazole hybrids 6a-o were prepared and characterized. The in vitro antimycobacterial activity revealed that compound 6a experienced promising antimycobacterial activity against Mycobactrium smegmatis compared with the reference isoniazide (INH). Additionally, compound 6a exhibited broad spectrum antibacterial activity against all the tested strains either Gram-positive or Gram-negative bacteria compared with the reference ciprofloxacin. Also, compounds 6g and 6i displayed considerable antifungal activity compared with the reference ketoconazole. DNA cleavage assay of the highly active compounds 6c and 6h showed a good correlation between the Mycobactrium cleaved DNA gyrase assay and their in vitro antimycobactrial activity. Moreover, molecular modeling studies were done for the designed ciprofloxacin derivatives to predict their binding modes towards Topoisomerase II enzyme (PDB: 5bs8).     

Reaction of pyridoxal 5'-phosphate with Escherichia coli CoA transferase: evidence for an essential lysine residue.

The acetyl-CoA:acetoacetate CoA-transferase of Escherichia coli was reversibly inactivated by pyridoxal 5′-phosphate. The residual activity of the enzyme was dependent on the concentration of the modifying reagent to a concentration of 5 mm. The maximum level of inactivation was 89%. Kinetic and equilibrium analyses of inactivation were consistent with a two-step process (Chen and Engel, 1975, Biochem. J.149, 619) in which the extent of inactivation was limited by the ratio of first-order rate constants for the reversible formation of an inactive Schiff base of pyridoxal 5′-phosphate and the enzyme from a noncovalent, dissociable complex of the enzyme and modifier. The calculated minimum residual activity was in close agreement with the experimentally determined value. The conclusion that the loss of catalytic activity resulted from modification of a lysine residue at the active site was based on the following data, (a) After incubation with 5 mm pyridoxal 5′-phosphate, 3.95 mol of the reagent was incorporated per mole of free enzyme with 89% loss of activity, while 2.75 mol of pyridoxal 5′-phosphate was incorporated into the enzyme-CoA intermediate with a loss of 10% of catalytic activity; the intermediate was formed in the presence of acetoacetyl-CoA; (b) acid hydrolysis of the modified, reduced enzyme-CoA intermediate yielded a single fluorescent compound that was identified as N⁶-pyridoxyllysine by chromatography in two solvent systems; (c) the enzyme was also protected from inactivation by saturating concentrations of free CoA and ADP but not by adenosine. The results suggested that a lysine residue is involved in the electrostatic binding of the pyrophosphate group of CoA. Carboxylic acid substrate did not protect the enzyme from inactivation.     

Rational design,molecular docking and synthesis of novel homopiperazine linked imidazo[1,2-a]pyrimidine derivatives as potent cytotoxic and antimicrobial agents

Designed and synthesized novel homopiperazine linked imidazo[1,2-a]pyrimidine derivatives (10a–i, 11a–g, 12), and evaluated them for their in vitro cytotoxicity against HeLa cells (cervical cancer), A549 cells (lung cancer) cells, by MTT assay. Compound 12 (IC₅₀ = 4.14 µM) and compound 10c (IC₅₀ = 5.98 µM) were found to be 2.5 fold, and 1.74 fold more potent when compared with standard Etoposide (IC₅₀ = 10.44 µM), against A549 (lung cancer cells). Compound 12 also found to be 1.57 and 1.13 fold potent against DU145 (IC₅₀ = 6.24 µM) and HeLa (IC₅₀ = 6.54 µM), respectively when compared with Etoposide (DU145, IC₅₀ = 9.8 µM; HeLa, IC₅₀ = 7.43 µM). Compound 10f (IC₅₀ = 6.12 µM) was found to be 1.31 fold more potent than Etoposide (IC₅₀ = 7.43 µM) against HeLa cell lines.Moreover compounds 10a and 11a showed cytotoxicity at low micro-molar concentrations against A549 cells. Synthesized compounds were also evaluated for their antimicrobial activity by Cup plate diffusion method. Compounds 10c, 11b, 11d and 11f displayed remarkable antimicrobial activity relating to their standard drugs Gentamycin, Amphotericin B and Ampicillin. Significantly, compound 10c showed broad spectrum activity against tested microbial strains. All the designed compounds were well occupied the binding site of the colchicine and interacted with both α- and β-tubuline interface (PDB ID: 3E22), which demonstrates that synthesized compounds are promising tubulin inhibitors. Also, the synthesized compounds occupied the catalytic triad and adenine-binding site, in the active site of β-ketoacyl-acyl carrier protein synthase III enzyme (PDB ID: 1MZS). The molecular docking results provided the useful information for the future design of more potent inhibitors. These preliminary results convinced further investigation and modifications on synthesized compounds aiming towards the development of potential cytotoxic as well as antimicrobial agents.     

Substrate-mediated increased reactivity of a critical sulfhydryl group of crystals of cytoplasmic aspartate transaminase

The extramitochondrial isozyme of aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1) contains a cysteinyl residue (cysteine-390) which, in the presence of substrate, displays enhanced reactivity toward sulfhydryl reagents. To gain insight into the structural similarity of the enzyme in solution compared to its crystalline state and into the type of structural change induced by substrates, the reactivity of Cys-390 in the crystalline enzyme has been studied. The flat yellow plates, crystallized from polyethylene glycol, form spectroscopically detectable enzyme-substrate complexes (C. M. Metzler, D. E. Metzler, D. S. Martin, R. Newman, A. Arnone, and P. Rodgers, 1978, J. Biol. Chem. 253, 5251–5254). The crystals, both in the presence and absence of the substrate pair, glutamate and α-ketoglutarate, were treated with N-ethylmaleimide or N-ethyl[1-¹⁴C]maleimide and the extent of the reaction was monitored by the colorimetric sulfhydryl reaction with 5,5′-dithiobis-2-nitrobenzoic acid, by amino acid analysis, by radioactivity incorporated, and by the measurement of enzyme activity. A cysteine residue was modified only in the presence of substrate; the crystals remained undamaged. Since, any large conformational change in the enzyme would either be prevented by the crystalline lattice or would disrupt its integrity, it is concluded that the enhanced reactivity of cysteine-390 in the presence of substrates must be due to only a small local conformational change in the substrate binding region.